Chemoenzymatic Synthesis of Glycolic Acid

Abstract

A chemoenzymatic process for the production of high-purity glycolic acid has been demonstrated, starting with the reaction of formaldehyde and hydrogen cyanide to produce glycolonitrile in > 99 % yield and purity. The resulting aqueous glycolonitrile was used without further purification in a subsequent biocatalytic conversion of glycolonitrile to ammonium glycolate. A high-activity biocatalyst based on an Acidovorax facilis 72W nitrilase was developed, where protein engineering and optimized protein expression in an E. coli transformant host were used to improve microbial nitrilase specific activity by 33-fold compared to the wild-type strain. A biocatalyst productivity of > 1000 g glycolic acid/g dry cell weight was achieved using a glutaraldehyde/ polyethylenimine cross-linked carrageenan-immobilized E. coli MG1655 transformant expressing the A. facilis 72W nitrilase mutant, where 3.2 M ammonium glycolate was produced in consecutive batch reactions with biocatalyst recycle, or in a continuous stirred-tank reactor. Direct conversion of the unpurified ammonium glycolate product solution to highpurity aqueous glycolic acid was accomplished by fixed-bed ion exchange over a strong acid cation resin.

Conversion of Sterically Demanding α,α-Disubstituted Phenylacetonitriles by the Arylacetonitrilase from Pseudomonas fluorescens EBC191

“…The first synthetic applications for nitrilases have been established for aromatic nitrilases (especially by using the enzyme from Rhodococcus rhodochrous J1), and examples for the conversion of compounds such as 3-cyanopyridine to nicotinic acid or cyanopyrazine to pyrazinoic acid have been investigated (24,30). Subsequently, several biotransformation reactions which utilize aliphatic nitrilases or arylacetonitrilases were also described (35,52,53). Arylacetonitrilases are able to convert ␣-substituted phenylacetonitriles and are therefore interesting for the enantioselective synthesis of various ␣-substituted carboxylic acids (and amides).…”

Optimization of Biocatalyst Specific Activity for Glycolic Acid Production

“…A mineral medium with glucose, ammonia and 5 g/L yeast extract was used for cell culture, where the pH was maintained with ammonia titration, glucose was supplied first in batch and fed batch, and later in the run lactose was added as substrate and inducer. [1] Washing the cells removed ammonia (30-50 mM) from the culture and rendered the washed cells significantly more sensitive to GA treatment than cells treated directly with GA in a broth, but for practical reasons an inactivation method was developed using unwashed cultures that can be used directly in the fermentation vessel.…”

“…[1] A glutaraldehyde/polyethylenimine cross-linked carrageenan-immobilized E. coli MG1655 transformant expressing the Acidovorax facilis 72W nitrilase [2] having a Phe168Val mutation [3] was employed as biocatalyst, and a biocatalyst productivity of > 1000 g glycolic acid/g dry cell weight was achieved during the production of 3.2 M ammonium glycolate in either consecutive batch reactions with biocatalyst recycle or in a continuous stirred-tank reactor.…”

Nitrile-Degrading Bacteria Isolated from Compost

“…Consequently, nitrile-converting enzymes are of increasing industrial importance with respect to green chemistry. A constantly increasing number of nitrile-derived amides [e.g., acrylamides or carboxylic acids (e.g., glycolic acid)] are produced with these enzymes (Schmid et al, 2001;Panova et al, 2007). In addition, nitrilases can be used for the treatment of nitrile-polluted wastewater (Li et al, 2016) and other environmentally-friendly bioremediation processes (Gong et al, 2012).…”